BIOLOGICAL SIGNIFICANCE: NKG2D is an activating receptor found on several types of immune cells. Cells stressed by transformation or infection express proteins such as MIC-A on their surface that bind NKG2D, initiating an important cytotoxic immune response. A mutant MIC-A that interacts with NKG2D more avidly would be an improved in vitro reagent and a potential therapeutic agent that could activate natural killer cells against cancer in a cellular context, or, if soluble, could downregulate natural killer cell activation in graft-vs.-host disease (GVHD). Progress Report: In our laboratory over the past three years, undergraduate student researchers have cooperated to produce more than 25 MIC-A variants with one to five point mutations. The thermodynamics and kinetics of these variants binding to NKG2D were measured with surface plasmon resonance. They were also characterized using other biophysical techniques including circular dichroism and fluorescence. We found several double or triple mutant MIC-A molecules that bound NKG2D with affinity and on-rates enhanced by more than an order of magnitude, fulfilling all of our previous Specific Aims. Hypothesis: The affinity-enhancing MIC-A mutations primarily affect on-rates and do not appear to contact NKG2D when complexed. Direct contacts with NKG2D have not yet been altered and can now be targeted for optimization to stabilize the off-rate. We will attempt to enhance MIC-A--NKG2D affinity by at least another order of magnitude by improving contacts using three design strategies (contact optimization, peripheral electrostatic manipulation, and grafting contacts from MIC-B). Method: Undergraduate student researchers will test this hypothesis by using RosettaDesign to compare potential mutations. We will make mutant MIC-A molecules using site-directed mutagenesis and established protein purification techniques. We will study the impact of these mutations on NKG2D--MIC-A stability, entropy, enthalpy, and kinetics using surface plasmon resonance binding analysis. More specifically stabilized NKG2D--MIC-A complexes should be enthalpically stabilized, and optimization of contacts is expected to decrease off-rates only. Teaching Aims: Students will develop and test specific protein design hypotheses. They will then learn standard protein manipulation and production techniques in the course of discovering new relationships between protein-protein interface structure, design, and thermodynamics. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: When a cell is stressed by a virus or cancer, it places a protein named MIC-A on its surface that activates the immune system against the problem. In this project, undergraduate students will use computational and biochemical techniques to design new versions of MIC-A that will bind the immune system's receptors more tightly. The students will make new MIC-A molecules in the lab and test how tightly they bind receptors to find which ones bind immune receptors more tightly and could be used to send more effective signals and activate the immune system against cancer. [unreadable] [unreadable] [unreadable]